Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na+, Li+, or H+. In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na+ or Li+ and stimulated by equimolar concentrations of internal and external Na+ or Li+. Melibiose exchange is faster than efflux in the presence of H+ or Na+ and stimulated by an inwardly directed Na+ gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li+ stimulates efflux, and an outwardly directed Li+ gradient increases exchange, it is striking that internal and external Li+ with no gradient inhibits exchange. Furthermore, Trp -> dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl) aminoalkyl-1-thio-beta-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na+ or Li+, exhibits K-app(d) values of similar to 1 mM for melibiose. Na+ and Li+ compete for a common binding pocket with activation constants for FRET of similar to 1 mM, whereas Rb+ or Cs+ exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H+ in addition to Na+ and Li+. FRET studies also show symmetrical emission maximum at similar to 500 nm with MelB-ST in the presence of 2'-(N-dansyl) aminoalkyl-1-thio-beta-D-galactopyranoside and Na+, Li+, or H+, which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.